In genetic research, diagnosis or forensic investigations, the scarcity of genomic DNA can be a severely limiting factor on the type and quantity of genetic tests that can be performed on a sample. One approach designed to overcome this problem is whole genome amplification. The objective is to amplify a limited DNA sample in a non-specific manner in order to generate a new sample that is indistinguishable from the original but with a higher DNA concentration. The aim of a typical whole genome amplification technique would be to amplify a sample up to a level sufficient to perform multiple tests and archiving procedures, while maintaining an accurate representation of the original sequence.
The first whole genome amplification methods were described in 1992, and were based on the principles of the polymerase chain reaction (PCR). Zhang and coworkers (Zhang et al. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5847-5851) developed the primer extension PCR technique (PEP) and Telenius and collaborators (Telenius et al., Genomics 1992, 13, 718-725) designed the degenerate oligonucleotide-primed PCR method (DOP-PCR).
PEP involves a high number of PCR cycles using Taq polymerase and 15 base random primers that anneal at a low stringency temperature. Although the PEP protocol has been improved in different ways, it still results in incomplete genome coverage, failing to amplify certain sequences such as repeats. Failure to prime and amplify regions containing repeats may lead to incomplete representation of a whole genome because consistent primer coverage across the length of the genome is required for complete representation of the genome. This method also has limited efficiency on very small samples (such as single cells). Moreover, the use of Taq polymerase is optimized for a maximal product length of about 3 kb. DOP-PCR is a method which uses Taq polymerase and semi-degenerate oligonucleotides that bind at a low annealing temperature at approximately one million sites within the human genome. The first cycles are followed by a large number of cycles with a higher annealing temperature, allowing only for the amplification of the fragments that were tagged in the first step. This leads to incomplete representation of a whole genome. DOP-PCR generates, like PEP, fragments that are in average 400-500 bp, with a maximum size of 3 kb, although fragments up to 10 kb have been reported. On the other hand, as noted for PEP, a low input of genomic DNA (less than 1 ng) decreases the fidelity and the genome coverage (Kittler et al. Anal. Biochem. 2002, 300, 237-244).
Multiple displacement amplification (MDA, is a non-PCR-based isothermal method based on the annealing of random hexamers to denatured DNA, followed by strand-displacement synthesis at constant temperature (Blanco et al. J. Biol. Chem. 1989, 264, 8935-8940). It has been applied to samples with small quantities of genomic DNA, leading to the synthesis of high molecular weight DNA with limited sequence representation bias (Lizardi et al. Nature Genetics 1998, 19, 225-232; Dean et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5261-5266). As DNA is synthesized by strand displacement, a gradually increasing number of priming events occur, forming a network of hyper-branched DNA structures. The reaction can be catalyzed by enzymes such as the Phi29 DNA polymerase or the large fragment of the Bst DNA polymerase. The Phi29 DNA polymerase possesses a proofreading activity resulting in error rates 100 times lower than Taq polymerase (Lasken et al. Trends Biotech. 2003, 21, 531-535).
The methods described above generally do not successfully amplify DNA samples when the quantity of template DNA being amplified is below the level of one 1 nanogram (ng). Problems encountered during such amplification attempts include, for example, poor representation of the original template DNA in the amplified product (Dean et al. Proc. Natl. Acad. Sci U.S.A. 2002, 99, 5261-5266) and competing amplification of non-template DNA (Lage et al. Genome Research 2003, 13, 294-307).
It has been established that a problem encountered when small amounts of template are amplified using Phi29 DNA polymerase is that “background” DNA synthesis usually occurs when template is omitted, or at low template concentrations. Reducing the reaction volume while keeping the amount of template fixed increases the template concentration, results in a suppression of background synthesis (Hutchinson, C. A. III et al., Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17332-17336). Another issue is poor representation and balance of the DNA of interest following whole genome amplification (WGA).
There remains a long felt need for methods and kits for performing whole genome amplification reactions on small quantities of DNA than maintains the genetic balance and representation of the original sample. The present invention satisfies this need.